Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane.

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the 'cold' synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.     

Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein

Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin). This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the salt-extract of microsomal vesicles (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles. Likewise, polysome binding to the microsomal membrane was largely abolished when beta-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides. The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions.     

Mechanism of compartmentation of secretory proteins: transport of exocrine pancreatic proteins across the microsomal membrane

The mechanism by which secretory proteins are segregated within the cisternal space of microsomal vesicles was studied using dog pancreas mRNA which directs the synthesis of 14 well-characterized nonglycosylated pancreatic exocrine proteins. In the absence of microsomal membranes, each of the proteins was synthesized as larger polypeptide chains (presecretory proteins). 1,000-2,000 daltons larger than their authentic counterparts as judged by polyacrylamide gel electrophoresis in SDS. Conditions optimal for the study of reconstituted rough microsomes in the reticulocyte lysate system were examined in detail using mRNA and microsomal membranes isolated from dog pancreas. Functional reconstitution of rough microsomes was considerably more efficient in the presence of micrococcal nuclease- treated membranes than in the presence of EDTA-treated membranes. Analysis for segregation of nascent secretory proteins by microsomal vesicles, using post-translational incubation in the presence of trypsin and chymotrypsin, 50 μg/ml each, was shown to be inadequate, because of the disruption of vesicles by protease activity. Addition of 1-3 mM tetracaine or 1 mM dibucaine stabilized microsomal membranes incubated in the presence of trypsin and chymotrypsin at either 0 degrees or 22 degrees C. Each of the pancreatic presecretory proteins studied was correctly processed to authentic secretory proteins by nuclease-treated microsomal membranes, as judged by both one-dimensional and two-dimensional gel electophoresis. Post-translational addition of membranes did not result in either segregation or processing of nascent polypeptide chains. Post- translational proteolysis, carried out in the presence of 3 mM tetracaine, indicated that each of the 14 characterized dog pancreas secretory proteins was quantitatively segregated by nuclease-treated microsomal vesicles. Segregation of nascent secretory proteins was irreversible, since radioactive amylase, as well as the other labeled secretory proteins, remained quantitatively sequestered in microsomal vesicles during a 90-min incubation at 22 degrees C after the cessation of protein synthesis. Studies employing synchronized protein synthesis and delayed addition of membranes indicated that all pancreatic presecretory proteins contain amino terminal peptide extensions. These peptide extensions are shown to mediate the cotranslational binding of presecretory proteins to microsomal membranes and the transport of nascent secretory proteins to the vesicular space. The maximum chain lengths which, during synthesis, allow segregation of nascent polypeptide chains varied between 61 (pretrypsinogen 2 + 3) and 88 (preprocarboxypeptidase A1) amino acid residues among dog pancreas presecretory proteins. Reconstitution studies using homologous and heterologous mixtures of mRNA (dog, guinea pig, and rat pancreas; rat liver) and micrococcal nuclease-treated microsomal membranes (dog, guinea pig, and rat liver; dog pancreas), in the presence of placental ribonuclease inhibitor, suggest that the translocation mechanism described is common to the rough endoplasmic reticulum of all mammalian tissues.     

Nascent chicken ovalbumin contains the functional equivalent of a signal sequence

Highly purified mRNA for chicken ovalbumin has been translated in a cell-free protein synthesizing system from rabbit reticulocytes in the presence or absence of EDTA-stripped microsomal membranes from dog pancreas. Nascent--but not completed--ovalbumin was transferred across the microsomal membrane, as demonstrated by cotranslational core glycosylation of ovalbumin nascent chains, by resistance to posttranslational proteolysis of only the glycosylated ovalbumin chains, and by cosedimentation with the membrane of exclusively the glycosylated form. Furthermore, nascent chains of bovine prolactin were observed to compete with nascent ovalbumin for transfer across the microsomal membrane. However, no competition for membrane sites was observed between nascent chains of rabbit globin and either nascent ovalbumin or prolactin. We interpret these results to suggest that nascent ovalbumin contains the functional equivalent of a signal sequence for transfer across membranes, and that membrane components involved in the segregation of secretory proteins with cleaved signal sequences also function in the segregation of ovalbumin.     

Regulation of signal peptidase by phospholipids in membrane: characterization of phospholipid bilayer incorporated Escherichia coli signal peptidase

Prokaryotic signal peptidases are membrane-bound enzymes. They cleave signal peptides from precursors of secretary proteins. To study the enzyme in its natural environment, which is phospholipid bilayers, we developed a method that allows us effectively to incorporate full-length Escherichia coli signal peptidase I into phospholipid vesicles. The membrane-bound signal peptidase showed high activity on a designed substrate. The autolysis site of the enzyme is separated from its catalytic site in vesicles by the lipid bilayer, resulting in a dramatic decrease of the autolysis rate. Phosphotidylethanolamine, which is the most abundant lipid in Escherichia coli inner membrane, is required to maintain activity of the membrane-incorporated signal peptidase. The maximal activity is achieved at about 55% phosphotidylethanolamine. Negatively charged lipids, which are also abundant in Escherichia coli inner membrane, enhances the activity of the enzyme too. Its mechanism, however, cannot be fully explained by its ability to increase the affinity of the substrate to the membrane. A reaction mechanism was developed based on the observation that cleavage only takes place when the enzyme and the substrate are bound to the same vesicle. Accordingly, a kinetic analysis is presented to explain some of the unique features of phospholipid vesicles incorporated signal peptidase, including the effect of lipid concentration and substrate-vesicle interaction.     

A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.     

Demonstration of post-translational secretion of human placental lactogen by a mammalian in vitro translation system

This study demonstrates the post-translational translocation across the rough endoplasmic reticular membrane of a mammalian secretory protein, human preplacental lactogen. In the rabbit reticulocyte lysate, human preplacental lactogen biosynthesis is arrested by addition of cycloheximide prior to supplementation with dog pancreatic microsomal membranes, which have previously been shown to translocate and process nascent secretory proteins in a cotranslational manner. Twenty-five percent of the precursor protein is consistently converted to its mature form under these post-translational conditions. The resulting mature hormone is resistant to proteolytic degradation by added proteases, thus indicating that it is translocated across the microsomal membrane and sequestered within the lumenal space of the microsomal vesicles. Approximately one-half of the precursor protein synthesized is associated with the ribosomes. Only the ribosome-associated fraction is secreted in this in vitro system, suggesting that the process of post-translational secretion requires ribosomes for protein interaction with the elements of a subcellular secretory apparatus.     

Phospholipase D Stimulates Release of Nascent Secretory Vesicles from the trans-Golgi Network

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.     

Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted

The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.     

Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor

The signal recognition particle (SRP)-mediated elongation arrest of the synthesis of nascent secretory proteins can be released by salt- extracted rough microsomal membranes (Walter, P., and G. Blobel, 1981, J. Cell Biol, 91:557-561). Both the arrest-releasing activity and the signal peptidase activity were solubilized from rough microsomal membranes using the nonionic detergent Nikkol in conjunction with 250 mM KOAc. Chromatography of this extract on SRP-Sepharose separated the arrest-releasing activity from the signal peptidase activity. Further purification of the arrest-releasing activity using sucrose gradient centrifugation allowed the identification of a 72,000-dalton polypeptide as the protein responsible for the activity. Based upon its affinity for SRP, we refer to the 72,000-dalton protein as the SRP receptor. A 60,000-dalton protein fragment (Meyer, D. I., and B. Dobberstein, 1980, J. Cell Biol., 87:503-508) that had been shown previously to reconstitute the translocation activity of protease- digested membranes, was shown here by peptide mapping and immunological criteria to be derived from the SRP receptor. Findings that are in part similar, and in part different from these reported here and in our preceding paper were made independently (Meyer, D. I., E. Krause, and B. Dobberstein, 1982, Nature (Lond.). 297:647-650) and the term "docking protein" was proposed for the SRP receptor. A lower membrane content of both SRP and the SRP receptor than that of membrane bound ribosomes suggests that the SRP-SRP receptor interaction may exist transiently during the formation of a ribosome-membrane junction and during translocation.     

Evidence for a tubulin-containing lipid-protein structural complex in ciliary membranes

The proteins and lipids of the scallop gill ciliary membrane may be reassociated through several cycles of detergent solubilization, detergent removal, and freeze-thaw, without significant change in overall protein composition. Membrane proteins and lipids reassociate to form vesicles of uniform, discrete density classes under a variety of reassociation conditions involving detergent removal and concentration. Freed of the solubilizing detergent during equilibrium centrifugation, a protein-lipid complex equilibrates to a position on a sucrose density gradient characteristic of the original membrane density. When axonemal tubulin is solubilized by dialysis, mixed with 2:1 lecithin/cholesterol dissolved in Nonidet P-40, freed of detergent, and reconstituted by freeze-thaw, vesicles of a density essentially equal to pure lipid result. If the lipid fraction is derived through chloroform-methanol extraction of natural ciliary membranes, a moderate increase in density occurs upon reconstitution, but the protein is adsorbed and most is removed by a simple low ionic strength wash, in contrast to vesicles reconstituted from membrane proteins where even high salt extraction causes no loss of protein. The proteins of the ciliary membrane dissolve with constant composition, regardless of the type, concentration, or efficiency of detergent. Analytical ultracentrifugation demonstrates that monodisperse mixed micelles form at high detergent concentrations, but that membranes are dispersed to large sedimentable aggregates by Nonidet P-40 even at several times the critical micelle concentration, which suggests reasons for the efficacy of certain detergent for the production of ATP-reactivatable cell models. In extracts freed of detergent, structured polydisperse particles, but not membrane vesicles, are seen in negative staining; vesicles form upon concentration of the extract. Membrane tubulin is not in a form that will freely undergo electrophoresis, even in the presence of detergent above the critical micelle concentration. All chromatographic attempts to separate membrane tubulin from other membrane proteins have failed; lipid and protein are excluded together by gel filtration in the presence of high concentrations of detergent. These observations support the idea that a relatively stable lipid-protein complex exists in the ciliary membrane and that in this complex membrane tubulin is tightly associated with lipids and with a number of other proteins.     

Properties of rat liver signal peptidase reconstituted into liposomes

EDTA/KCl- or pyrophosphate-treated rough microsomes of rat liver clearly showed the co-translational cleavage of pre-human placental lactogen and translocation of the product into membrane vesicles. The signal peptidase fraction was isolated by chromatography on Sephacryl S-300 of deoxycholate-treated membranes and reconstituted into liposomes by dialysis or by the Biobeads SM-2 method. Assay of the signal peptidase activity was performed with pre-human placental lactogen synthesized by the reticulocyte lysate system programmed with human placental lactogen mRNA. The signal peptidase reconstituted into liposomes showed stable activity over the temperature range of 0 to 45 degrees C; in contrast, the detergent-solubilized signal peptidase of dog pancreatic membranes was completely inactivated at the unusually low temperature of 37 degrees C. It was shown that this inactivation was due to the presence of detergent. Signal peptidase from rat liver was insensitive to a variety of protease inhibitors, like the enzyme from dog pancreas, but differed from the latter in being inhibited by chymostatin and TPCK.     

Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins

Fluorescence resonance energy transfer measurements reveal that a transmembrane sequence within a nascent membrane protein folds into a compact conformation near the peptidyltransferase center and remains folded as the sequence moves through a membrane bound ribosome into the translocon. This compact conformation is compatible with an alpha helix because nearly the same energy transfer efficiency was observed when the transmembrane sequence was integrated into the lipid bilayer. Since the transmembrane sequence unfolds upon emerging from a free ribosome, this nascent chain folding is ribosome induced and stabilized. In contrast, a nascent secretory protein is in an extended conformation in the exit tunnel. Furthermore, two ribosomal proteins photo-crosslink to nascent membrane but not secretory proteins. These interactions coincide with the previously described sequential closing and opening of the two ends of the aqueous translocon pore, thereby suggesting that ribosomal recognition of nascent chain folding controls the operational mode of the translocon at the ER membrane.     

Reconstitution of purified dipeptidyl peptidase IV. A comparison with aminopeptidase N with respect to morphology and influence of anchoring peptide on function

The pig small intestinal dipeptidyl peptidase IV was asymmetrically integrated into egg phosphatidylcholine and microvillar lipid vesicles prepared by a beta-octylglucoside dialysis method. The enzyme molecules appeared dumbell-shaped ((11.0-11.5) X (5.0-5.5)nm) and were separated from the liposomal membrane by a stain-filled gap of about 2.5 nm, representing the 'junctional segment'. The influence of lipid bilayer and detergents on the kinetic parameters of amphiphilic and hydrophilic forms of aminopeptidase N and dipeptidyl peptidase IV was studied. Since the lipid bilayer and detergents, which interact only with the anchoring root, had no crucial effect on the kinetic parameters of the different forms of the enzymes, it is concluded that the anchoring roots exert little effect on the catalytic domain of the stalked integral membrane proteins.     

Supported double membranes

Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre- and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin–streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.     

A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.     

Flippases and vesicle-mediated protein transport

The best-understood mechanisms for generating transport vesicles in the secretory and endocytic pathways involve the localized assembly of cytosolic coat proteins such as clathrin, coat protein complex (COP)I and COPII onto membranes. These coat proteins can deform membranes by themselves, but accessory proteins might help to generate the tight curvature needed to form a vesicle. Enzymes that pump phospholipid from one leaflet of the bilayer to the other (flippases) can deform membranes by creating an imbalance in the phospholipid number between the two leaflets. Recent studies describe a requirement for the yeast Drs2p family of P-type ATPases in both phospholipid translocation and protein transport in the secretory and endocytic pathways. This indicates that flippases work with coat proteins to form vesicles.     

ER-to-Golgi transport: COP I and COP II function (Review)

COP I and COP II coat proteins direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells. These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles. COP II proteins are required for selective export of newly synthesized proteins from the endoplasmic reticulum (ER). COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER. Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex.     

Nascent Lep inserts into the Escherichia coli inner membrane in the vicinity of YidC, SecY and SecA

Targeting and assembly of the Escherichia coli inner membrane protein leader peptidase (Lep) was studied using a homologous in vitro targeting/translocation assay. Assembly of full-length Lep was efficient in the co-translational presence of membrane vesicles and hardly occurred when membranes were added post-translationally. This is consistent with the signal recognition particle-dependent targeting of Lep. Crosslinking experiments showed that the hydrophilic region P1 of nascent membrane-inserted Lep 100-mer was in the vicinity of SecA and SecY, whereas the first transmembrane domain H1 was in the vicinity of YidC. These results suggested that YidC, together with the Sec translocase, functions in the assembly of Lep. YidC might be a more generic component in the assembly of inner membrane proteins.     

Amino acid sequences of transport peptides associated with canine exocrine pancreatic proteins

Using microsequencing techniques and proteins labeled in vitro with tritiated amino acids we have obtained the following NH2-terminal sequences for six canine pancreatic presecretory proteins: pretrypsinogen 1, pretrypsinogen 2+3, prechymotrypsinogen 2, preproelastase1, preporcarboxypeptidase A1, preamylase. Points of cleavage by the transport peptidase, indicated by the vertical arrows, were located from sequences of authentic products synthesized in the presence of membranes of the rough endoplasmic reticulum. All of the identified residues in the pancreatic transport peptides are hydrophobic. Predictions of secondary structure were calculated for each of the transport peptides. The data indicated neither a common primary of secondary structure which could be interpreted as the signal for functional binding of the nascent presecretory protein to the rough endoplasmic reticulum membrane. These findings suggest that the initial interaction with the membrane or membrane receptor may depend in part, on the hydrophobic nature of the transport peptides. Five of the presecretory proteins showed a region with a high probability of forming a beta-turn immediately following the cleavage point. This feature may give the nascent peptide a region of flexibility that would facilitate both its insertion as a loop structure into the membrane and its cleavage by the transport peptidase. The sequences of authentic secretory products derived from a variety of pancreatic tissues suggest that hydrophilic residues are required immediately following the cleavage point in order to allow translocation of the nascent polypeptide chains across the membrane.